Methods using eosinophil-specific apoptosis inducer

ABSTRACT

The present invention provides an apoptosis inducer and a therapeutic agent for eosinophilic diseases which comprises, as an active ingredient, an antibody which reacts specifically with eosinophils and induces apoptosis of eosinophils; and a method for inducing eosinophil apoptosis using the antibody, and a method for specifically reducing or removing eosinophils in peripheral blood or tissues using the antibody.

This is the national stage under 37 CFR §3.71 of InternationalApplication No. PCT/JP01/01077 filed Feb. 15, 2001 which in turn claimspriority of Japanese application number 2000-036671 filed on Feb. 15,2000.

TECHNICAL FIELD

The present invention relates to an apoptosis inducer and a therapeuticagent for eosinophil associated diseases which comprises, as an activeingredient, an antibody which reacts specifically with eosinophils andinduces apoptosis of eosinophils. The present invention also relates toa method for specifically inducing eosinophil apoptosis using theantibody, and a method for specifically reducing or removing eosinophilsin peripheral blood or a tissue using the antibody.

BACKGROUND ART

Eosinophils are implicated in various diseases including allergicdiseases, and are thought to play an important role in generatingmorbidity of allergic diseases such as chronic bronchial asthma andatopic dermatitis [Adv. Immunol., 39, 177(1986), Immunol. Today, 13,501(1992)].

In addition to the above diseases, eosinophils are also implicated indiseases generally referred to as hypereosinophilic syndrome (HES), suchas eosinophilia, eosinophilic enterogastritis, eosinophilic leukemia,eosinophilic granuloma and Kimura's disease [Ann. Intern. Med., 97, 78(1982)].

Eosinophilic granuloma is nonneoplastic cryptogenic lesion, which is anosteolytic and focal, and is known to be associated with remarkabletissue cosinophulia [U.S. Armed Forces Med. J., 2, 1085 (1951)].According to the registry of bone tumor patients in Japan (1972-1984),379 out of 404 bone tumor patients (93.8%) suffered from eosinophilicgranuloma. Hence, in addition to inflammatory diseases, such as allergy,eosinophils can cause other various diseases.

Interleukin-5 (hereinafter referred to as IL-5), interleukin-3(hereinafter referred to as IL-3) and granulocyte-macrophagecolony-stimulating factor (hereinafter referred to as GM-CSF), which aremembers of cytokine family, are involved in regulating thedifferentiation, proliferation and activation of eosinophils. Of thesecytokines, IL-5 is known to act specifically on eosinophils andspecifically induce the terminal differentiation [Proc. Natl. Acad. Sci.U.S.A., 85, 2288 (1988)].

An anti-IL-5 antibody has been developed as an anti-inflammatory agent.A humanized anti-IL-5 antibody, SB-240563 (Smithkline Beecham), iseffective in reducing the number of eosinophils in peripheral blood ofmild asthma patients (100th Annual Meetings of the American Society forClinical Pharmacology and Therapeutics, March/1999). Moreover, ahumanized anti-IL-5 antibody, Sch-55700 (CDP-835)(Scherring-Plough/Celltech) is known to inhibit lung eosinophiliainduced by antigens in allergic monkey models [Arzneimittel-Forschung,49, 779 (1999)].

In vitro, IL-3 and/or GM-CSF can activate eosinophils or prolong theirsurvival [J. Clin. Invest., 81, 1986 (1988)]. Further, IL-3 and/orGM-CSF acts also predominantly on the induction of immature eosinophilsfrom myeloid stem cells [Blood, 76, 1956 (1990)]. Furthermore,chemokines such as eotaxin and RANTES (regulated on activation normalT-cell expressed and secreted), induce the chemotaxis of eosinophils toinflamed site [Clin. Exp. Allergy, 26, 1005 (1996)]. Stem cell factors(hereinafter referred to as SCF) are involved in the accumulation ofeosinophils to lung in allergic bronchitis. In addition to IL-5, thereare many factors affecting function of eosinophils.

Eosinophils are divided into subgroups, normodense eosinophils andhypodense eosinophils. Eosinophils have been shown to be hypodenseeosinophils upon activation [Immunology, 47, 531 (1982)]. Hypodenseeosinophils are also referred to as activated eosinophils. It has beenreported that a qualitative change occurs in addition to a quantitativechange in eosinophils in the peripheral blood of an HES patients [Clin.Exp. Immunol., 24, 423 (1976)]. Activated eosinophils have beenimplicated in the severity of HES symptom [Am. J. Cardiol., 52, 321(1983)]. Aside from HES patients, activated eosinophils have been alsofound in the peripheral blood, and in bronchoalveolar lavage fluid(BALF) of a patient with bronchial asthma [Am. Rev. Respir. Dis, 132,981 (1985)]. Various receptors, such as those of cytokines, areexpressed on activated eosinophils (hypodense eosinophils) [J. Immunol.,142, 4416 (1989)]. Compared to normodense eosinophils, these hypodenseeosinophils show elevated sensitivities against IL-5 [Clin. Exp.Immunol., 85, 312 (1991); J. Exp. Med., 172, 1347 (1990)].

The above-mentioned activated eosinophils are also known to survive invitro without the cytokines inducing in the differentiation andproliferation of eosinophils [J. Exp. Med., 170, 343(1989)]. Thus, theproperties of activated eosinophils are similar to those of eosinophilswhich infiltrate tissues, such as alveoli [Int. Arch. Allergy Immunol.,120, 91 (1999)]. A detailed explanation of why activated eosinophilsbecome cytokine-independent remains unknown, however, theirdegranulation and prolonged survival are likely to be induced by variousvital functional molecules other than IL-5.

Substances having inhibition activity on cytokines or chemokines thatare involved in the differentiation or proliferation of eosinophils havebeen considered as agents that inhibit the eosinophil functions.However, in most cases these agents do not act on cytokine-independenteosinophils that have been activated and infiltrated into inflamedareas. Hence, eosinophil-specific inhibition and the induction ofcellular death of activated eosinophils are necessary to inhibit thefunctions of any eosinophil.

However, no anti-inflammatory agent, so far, has been known to induceapoptosis of activated eosinophils.

Current major treatment for patients with eosinophilic diseases consistsof administration with steroid. However, steroid administration is oftenassociated with side effects. Specifically, the treatment has some otherproblems, such that patient's pathological condition may return to theoriginal state when steroid administration is discontinued, andprolonged steroid administration may induce steroid resistance. As fornow, it is difficult to inhibit the eosinophilia and there exists no wayother than the symptomatic treatment thereof.

SUMMARY OF THE INVENTION

The development of a clinically more effective treatment having lowerside effects has been long awaited for the treatment of inflammatorydiseases, such as chronic bronchial asthma, and eosinophil associateddiseases, such as eosinophilic granuloma.

Inventors of the present invention have found that humaneosinophil-specific apoptosis induced by an anti-IL-5 receptor α-chainantibody with an Fc region of the human IgG1 subclass as disclosed inWO97/10354 is mediated by antibody-dependent cellular cytotoxicity.Since apoptosis of eosinophils mediated by antibody-dependent cellularcytotoxicity does not cause the release of cytotoxic proteins, reducedside effects can be expected. In addition, the inventors of the presentinvention have shown that the antibody induces apoptosis ofIL-5-independent activated eosinophils, suggesting that the antibody isuseful in the treatment for eosinophilic diseases.

Specifically, the present invention relates to the following (1) to(20):

(1) An apoptosis inducer, comprising an antibody which reactsspecifically with an eosinophil and induces apoptosis of the eosinophilas an active ingredient.

(2) The apoptosis inducer of above mentioned (1), wherein theapoptosis-inducing antibody has antibody-dependent cellularcytotoxicity.

(3) The apoptosis inducer of above mentioned (1) or (2), wherein theantibody which reacts specifically with an eosinophil is an anti-humaninterleukin-5 receptor α-chain monoclonal antibody.

(4) The apoptosis inducer of above mentioned (3), wherein the anti-humaninterleukin-5 receptor α-chain monoclonal antibody is produced by ananimal cell.

(5) The apoptosis inducer of above mentioned (3), wherein the anti-humaninterleukin-5 receptor α-chain monoclonal antibody is produced by atransformant KM8399 (FERM BP-5648).

(6) A therapeutic agent for eosinophilic diseases, comprising anantibody which reacts specifically with an eosinophil and inducesapoptosis of the eosinophil as an active ingredient.

(7) The therapeutic agent for eosinophilic diseases of above mentioned(6), wherein the apoptosis-inducing antibody has cellular cytotoxicity.

(8) The therapeutic agent for eosinophilic diseases of above mentioned(6) or (7), wherein the antibody which reacts specifically with aneosinophil is an anti-human interleukin-5 receptor α-chain monoclonalantibody.

(9) The therapeutic agent for eosinophilic diseases of above mentioned(8), wherein the anti-human interleukin-5 receptor α-chain monoclonalantibody is produced by an animal cell.

(10) The therapeutic agent for eosinophilic diseases of above mentioned(8), wherein the anti-human interleukin-5 receptor α-chain monoclonalantibody is produced by the transformant KM8399 (FERM BP-5648).

(11) A method for specifically inducing apoptosis of an eosinophil usingan antibody which reacts specifically with an eosinophil and inducesapoptosis of the eosinophil.

(12) The method of above mentioned (11), wherein the apoptosis-inducingantibody has cellular cytotoxicity.

(13) The method of above mentioned (11) or (12), wherein the antibodywhich reacts specifically with an eosinophil is an anti-humaninterleukin-5 receptor α-chain monoclonal antibody.

(14) The method of above mentioned (13), wherein the anti-humaninterleukin-5 receptor α-chain monoclonal antibody is produced by ananimal cell.

(15) The method of above mentioned (13), wherein the anti-humaninterleukin-5 receptor α-chain monoclonal antibody is produced by thetransformant KM8399 (FERM BP-5648).

(16) A method for specifically reducing or removing eosinophils inperipheral blood or in a tissue infiltrated with eosinophils using anantibody which specifically reacts to an eosinophil and inducesapoptosis of the eosinophil.

(17) The method of above mentioned (16), wherein the apoptosis-inducingantibody has antibody-dependent cellular cytotoxicity.

(18) The method of above mentioned (16) or (17) for specificallyreducing or removing eosinophils, wherein the antibody which reactsspecifically with an eosinophil is an anti-human interleukin-5 receptorα-chain monoclonal antibody.

(19) The method of above mentioned (18), wherein the anti-humaninterleukin-5 receptor α-chain monoclonal antibody is produced by ananimal cell.

(20) The method of above mentioned (18), wherein the anti-humaninterleukin-5 receptor α-chain monoclonal antibody is produced by thetransformant KM8399 (FERM BP-5648).

As the antibody used for the present invention, any antibody whichreacts specifically to an eosinophil and induce apoptosis of theeosinophil can be used.

Examples of antibodies which react specifically to eosinophils includeantibodies against receptors expressed on the surfaces of eosinophils.Examples of antibodies against receptors expressed on the surfaces ofeosinophils include anti-human interleukin-5 receptor β-chainantibodies, anti-human interleukin-3 receptor antibodies, anti-humanmonocyte/macrophage colony-stimulating factor receptor antibodies, andanti-human interleukin-5 receptor α-chain (hereinafter, referred to ashIL-5R α) antibodies. The anti-hIL-5R α antibody is preferred.

Examples of antibodies which induce apoptosis of eosinophils includeantibodies having activity to inhibit signal transduction involved inthe differentiation or proliferation of eosinophils, and antibodieshaving cellular cytotoxicity. Antibodies having cellular cytotoxicityare preferred, in order to induce apoptosis of any eosinophil asdescribed below.

Hence, examples of antibodies capable of reacting specifically witheosinophils and inducing apoptosis of the eosinophils include antibodiesagainst receptors expressed on the surfaces of eosinophils, which haveantibody-dependent cellular cytotoxicity, and preferably, anti-hIL-5R αantibodies which have antibody-dependent cellular cytotoxicity. Otherexamples of such antibodies include antibodies against receptorsexpressed on the surfaces of eosinophils, which are produced by animalcell lines, such as CHO cells, YB2/3.0-Ag20 cells, SP2/0-AG14 cells andNS0 cells, and preferably, anti-hIL-5R α antibodies which are alsoproduced by animal cell lines. Further examples of such antibodiesinclude human IgG1 type antibodies against receptors expressed on thesurfaces of eosinophils, and preferably, human IgG1 type anti-hIL-5R αantibodies. An example is an anti-hIL-5R α human CDR-grafted antibodyKM8399 produced by a transformant KM8399 (FERM BP-5648).

The anti-IL-5 receptor α-chain antibody can be produced by a methoddescribed in WO97/10354.

Apoptosis of eosinophils induced by the above antibody can be confirmedby the following method.

1. Isolation of Eosinophil

(1) Isolation of Granulocyte from Peripheral Blood

Peripheral blood should be first treated with an anticoagulant toisolate the granulocytes from peripheral blood. Examples ofanticoagulants include heparin sodium, disodium EDTA and dipotassiumEDTA. Normally, 100 units of heparin sodium is used for 20 to 30 ml ofperipheral blood.

Peripheral blood is collected with a syringe containing ananticoagulant, superposed on a suitable isolation medium, andcentrifuged, thereby separating leukocytes into different cellpopulations, such as mononuclear cells, granulocytes and monocytes[Nature, 204, 793 (1964)].

Examples of media for separating peripheral blood-derived mononuclearcells (hereinafter referred to as PBMC) from granulocytes includeLymphoprep, Polymorphoprep (NYCOMED), Ficoll (Sigma) or the like.Further, isolation can also be performed using isotonic Percoll(Pharmacia) (0.15 M NaCl) adjusted to density of 1.085 to 1.088 by adensimeter. Centrifugation using the above isolation medium is alwaysperformed at room temperature.

(2) Isolation of Eosinophils From Granulocytes

Granulocytes separated in above mentioned (1) contain neutrophils andeosinophils, or may also contain erythrocytes. Erythrocytes can beremoved through hemolyzation by either one of the following methods:

The pellet of granulocytes containing erythrocytes in a centrifuge tubeis suspended in ice-cooled distilled water. After 30 seconds, anice-cooled 1/10 volume of isosmotic 10-fold concentration buffer isadded to stop hemolytic reaction. Centrifugation is carried out at 4° C.for 5 minutes at 400×g to remove the supernatant. Erythrocytes can beremoved by repeating the procedure a few times.

Alternatively, the pellet of granulocytes containing erythrocytes issuspended in an ice-cooled 0.2% NaCl solution. After 15 seconds, anequivalent volume of ice-cooled 1.6% NaCl solution is added to stop thehemolytic reaction, followed by centrifugation at 4° C. for 5 minutes at300×g, so that erythrocytes can also be removed [Clinical Immunology,29., (Suppl. 17), 41 1997].

Subsequent to removal of erythrocytes, neutrophils should be removed.

Neutrophils expressing CD16 antigen on their surface can be removed byperforming sorting.

First, granulocytes are incubated with mouse anti-CD 16 antibodies andthen sheep anti-mouse immunoglobulin antibody immobilized on theDynabead™ (DYNAL) is added. Using Magnetic bead concentrator MPC-1(DYNAL), Dynabead-bound CD 16 positive cells are captured to collect theremaining suspended cells, thereby isolating the eosinophils (Allergy,50, 34 (1995); Eur. J. Immunol., 24, 518 (1994); J. Immunol. Methods,122, 97 (1989)).

Neutrophils can also be separated from granulocytes by MACS™ system(Miltenyi) using anti-CD 16 antibody immobilization microbeads (J.Immunol. Methods, 165, 253 (1993), J. Immunol. Methods 127, 153 (1990)).

(3) Induction of Activated Eosinophil

Activated eosinophils can be obtained by culturing for a few days ofabove-mentioned (2) with IL-3[J. Clin. Invest., 81 1986 (1988)], or byco-culturing with PBMC for 2 days. Furthermore, blood collected from abody is centrifuged with cell isolation media of different densities, sothat activated eosinophils which have densities lower than the normallevels can be obtained [Clin. Exp. Immunol., 85, 312 (1991)].

The presence of activated eosinophils can be confirmed by the expressionof CD69 molecules [J. Exp. Med., 172, 701 (1990)].

(4) Method for Culturing Eosinophils

Eosinophils can be cultured in RPMI1640 media supplemented with 1% or10% fetal calf serum (hereinafter referred to as FCS), to which any oneof cytokines including IL-5, IL-3 and GM-CSF is added at a finalconcentration of 1 ng/ml under the air containing 5% CO₂ at 37° C.

2. Method for Inducing Apoptosis of Human Eosinophils by Antibodies

Inhibition of signal-transduction involved in the differentiation andproliferation of eosinophils causes normodense eosinophils to die.However, the inhibition of signal-transduction involved indifferentiation and proliferation of eosinophils is not enough to causehypodense eosinophils (activated eosinophils) to die. Hypodenseeosinophils cause effector function of antibody such ascomplement-dependent cytotoxicity (CDC), antibody-dependent cellularcytotoxicity (ADCC) or the like to die.

There are two types of cell death, necrosis and apoptosis. However, themechanism of action is yet to be elucidated.

Apoptosis can be induced by cytotoxic activity of antibody [CancerImunol. Immunother, 43, 220 (1996)]. However, the cellular cytotoxicitycauses not only apoptosis, but also necrosis.

Cell death of eosinophils induced by treating antibody used for thepresent invention having cellular cytotoxicity can be analyzed by thefollowing.

An example of a method for detecting necrotic cells involves stainingintracellular DNA with PI (Propidium Iodide) reagent; and an example ofa method for detecting apoptotic cells uses annexin V. Specifically,apoptotic cells can be evaluated by measuring cell surfacephosphatidylserine (hereinafter referred to as PS) with annexin V [J.Immunol. Methods, 217, 61 (1998)] as an indication as described in thefollowing method.

PS on the cell membrane is located on the side of cytoplasm in a livingcell. When apoptosis is induced, PS is exposed on the cell surfacewithin 1 hour. Accordingly, FITC-labeled annexin V which binds to PS ina calcium-dependent manner can detect the PS exposed apoptotic cells, sothat early apoptosis can be detected before the cell membrane is damaged[J. Exp. Med. 182, 1545 (1995)].

Double staining with annexin V-FITC and PI is preferred, because bindingof annexin V to cell membranes may also be observed in necrotic cells.Early apoptosis can be detected by the fact that it is stained withannexin V-FITC, but not with PI.

The antibody-dependent cellular cytotoxicity (hereinafter abbreviated asADCC) can be measured according to the method of 3 described later. Thusinduction of apoptosis in the target cells can be evaluated using theannexin V method.

3. Measurement of ADCC Activity

To measure ADCC activity, effector cells and target cells are used.

Examples of effector cells include natural killer (NK) cells, largegranular lymphocytes (LGL), and PBMC comprising NK and LGL, orleukocytes having Fc receptors on the cell surfaces, such asneutrophils, eosinophils and macrophages.

Effector cells can be isolated according to the method of abovementioned 1.

As the target cells, any cells which express, on the cell surfaces,antigens that antibodies to be evaluated can recognize can be used. Anexample of such a target cell is an eosinophil which expresses IL-5receptor on the cell surface.

Target cells are labeled with a reagent that enables detection ofcytolysis.

Examples of reagents for labeling include a radio-active substance suchas sodium chromate (Na₂ ⁵¹CrO₄, hereinafter referred to as ⁵¹Cr)[Immunology, 14, 181 (1968)], calcein-AM [J. Immunol. Methods, 172, 227(1994)], Europium [J. Immunol. Methods, 184, 29 (1995)] and ⁵¹Cr ispreferred.

When human peripheral blood eosinophils, which are terminallydifferentiated cells and have low labeling efficiency, are used astarget cells, the death of target cells should be detected by anothermethod after ADCC reaction. In this situation, cell death can bedetected by the method described in above mentioned 2.

4. Method for Specifically Reducing or Removing Eosinophils inPeripheral Blood or in Tissues Infiltrated with Eosinophils

Eosinophils can be specifically reduced or removed from peripheral bloodor tissues infiltrated with eosinophils using an apoptosis inducer whichcomprises, as an active ingredient, an antibody of the present inventionthat specifically reacts to the eosinophils and induces apoptosis of theeosinophils. Examples of such antibodies as an active ingredient includeanti-hIL-5R α-chain antibodies, or preferably anti-hIL-5R α antibodiesproduced by animal cells. For example, direct action of anti-hIL-5Rα-chain monoclonal antibodies KM8399 on peripheral blood or tissuesenables induction of eosinophil apoptosis, and reduction or removal ofeosinophils in peripheral blood or tissues infiltrated with eosinophils.

5. Form of Agent

The above-described apoptosis inducer or the therapeutic agent foreosinophilic diseases comprising, as an active ingredient, an antibodywhich specifically reacts to eosinophils and induces apoptosis of theeosinophils, may be solely administered as an agent. Normally, theinducer or the therapeutic agent is preferably provided aspharmaceutical preparations which are produced by mixing with one ormore pharmacologically acceptable carriers according to any method knownin the pharmaceutical technical field.

It is preferable to use an administration which is most effective incarrying out a treatment. Examples include oral administration andparenteral administration such as intraoral, bronchial, intrarectal,subcutaneous, intramuscular, intravenous administrations and the like.In an antibody-containing pharmaceutical formulation, intravenousadministration is preferrable.

Examples of dosage form include nebulae, capsules, tablets, granules,syrups, emulsions, suppositories, injection, an ointments, tapes and thelike.

Examples of formulation suitable for oral administration includeemulsions, syrups, capsules, tablets, powders, granules and the like.

Liquid preparations, such as emulsions and syrups, can be produced byusing as an additive, water; sugar, such as sucrose, sorbitol, fructoseetc.; glycol, such as polyethylene glycol, propylene glycol etc.; oil,such as sesami oil, olive oil, soybean oil etc.; antiseptic such asp-hydroxy benzoate ester etc.; flavoring, such as strawberry flavors,peppermint flavors; and the like.

Capsules, tablets, powders, granules or the like can be produced byusing as an additive, excipients such as lactose, glucose, sucrose,mannitol etc.; disintegrators, such as starch, sodium alginate etc.;lubricants, such as magnesium stearate, talc etc.; binders, such aspolyvinyl alcohol, hydroxypropylcellulose, gelatin etc.; surfactants,such as fatty acid ester etc.; plasticizers, such as glycerine etc; andthe like.

Examples of pharmaceutical preparations suitable for parenteraladministration include injectables, suppositories, nebulae and the like.

An injection is prepared by using a carrier or the like which comprisesa saline solution, a glucose solution, a mixture of both or the like.

A suppository is prepared by using a carrier, such as cacao butter,hydrogenated fat, carboxylic acid and the like.

A nebula is prepared by using the antibody preparation itself or using acarrier or the like which facilitates absorption by allowing thecompound to disperse as fine particles without stimulating the mouthcavity and bronchial mucous membrane of a recipient.

Examples of carriers include lactose, glycerine and the like.Preparations, such as aerosol and dry powder, can be used, depending onthe properties of the antibody and the carrier to be used. In addition,these parenteral preparations can be supplemented with componentsillustrated as additives for oral preparations.

The applied dose and the number of administration vary depending ontarget therapeutic effects, medication methods, treatment period, ageand body weight of the patient. Normally, 10 μg/kg to 8 mg/kg isadministered per day to an adult patient.

The term “eosinophil associated diseases” of the present inventionrefers to diseases caused by eosinophils, including allergic diseases,such as asthma bronchiale and atopic dermatitis; and hypereosinophilicsyndrome (HES), such as eosinophilia (e.g., eosinophilic pneumonia andsudden eosinophilia), eosinophilic enterogastritis, eosinophilicleukemia, eosinophilic granuloma and Kimura's disease.

The apoptosis inducer and the therapeutic agent for eosinophilicdiseases of the present invention can be used as the therapeutic agentfor the above eosinophil associated diseases.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the in vitro inhibitory effect on the IL-5-dependentsurvival of eosinophils isolated from peripheral blood. The verticalaxis indicates the positive % of annexin V resulting from the inhibitionof recombinant human IL-5 action (hereinafter referred to as rhIL-5),and the horizontal axis indicates each antibody. Human IgG1 was used asa negative control.

FIG. 2 shows the stainability of eosinophils fractions for each reagentafter reaction with antibodies. The vertical axis indicates eachreagent's positive % in the total number of eosinophils, and thehorizontal axis indicates the staining reagents. “Annexin V-FITCpositive cells” indicate apoptotic cells, and “PI positive cells”indicate necrotic cells.

FIG. 3 shows the specificity for cell of the apoptosis-inducing activityof KM8399. The vertical axis indicates the positive % of annexin V, andthe horizontal axis indicates each cell population.

FIG. 4 shows the apoptosis-inducing activity of KM8399 and TRFK5. Thevertical axis indicates the positive % of annexin V, and the horizontalaxis indicates the antibodies added.

FIG. 5 shows the dose dependency of the apoptosis-inducing activity ofKM8399. The vertical axis indicates the positive % of annexin V, and thehorizontal axis indicates the antibodies added.

FIG. 6 shows apoptosis-inducing activity in the presence of variouscytokines. The vertical axis indicates the positive % of annexin V, andthe horizontal axis indicates the cytokines added.

FIG. 7 shows the quantitatively determined values of eosinophilicgranular protein (EPO) released after ADCC reaction. The vertical axisindicates the proportions (%) of released EPO to the total EPO, and thehorizontal axis indicates the antibodies added.

FIG. 8 shows the quantitatively determined values of eosinophilicgranular protein (EDN) released after ADCC reaction. The vertical axisindicates the proportions (%) of released EDN to the total EDN, and thehorizontal axis indicates the types of antibodies added.

FIG. 9 shows the viability of eosinophils. The vertical axis indicatesdead cells (%), and the horizontal axis indicates the antibodies added.

FIG. 10 shows the increasement of CD16 positive cells after incubationwith KM8399. The vertical axis indicates the percentage of CD16 positivecells in the granulocytes, and the horizontal axis indicates antibody.

FIG. 11 shows the apoptosis-inducing activity on activated eosinophils.The vertical axis indicates the positive % of annexin V, and thehorizontal axis indicates antibody.

BEST MODE FOR CARRYING OUT THE INVENTION EXAMPLE 1 Isolation of HumanPBMC and Eosinophils

Sixty ml of normal human peripheral blood was collected using a syringecontaining 200U (200 μl) of a solution of heparin sodium for injection(Takeda Chemical Industries, Ltd.). The total volume was diluted twicewith the equivalent volume of saline (Otsuka Pharmaceutical Co., Ltd.)to obtain a final volume of 120 ml. Five ml of Lympho-prep (NYCOMED) wasapportioned to twelve 15 ml centrifuge tubes (SUMITOMO BAKELITE Co.,Ltd.), and then 10 ml of the diluted peripheral blood was superposedover each Lympho-prep, followed by centrifugation at 800×g for 20minutes at room temperature. A PBMC fraction between the plasma layerand the Lympho-prep layer was collected from all the centrifuge tubes,suspended in RPMI1640 media containing 1% FCS (GIBCO, hereinafterreferred to as 1% FCS-RPMI), and then washed twice by centrifugation at400×g for 5 minutes at 4° C., thereby preparing effector cells. Theportions other than the precipitation layers containing erythrocyteswere removed with an aspirator from the 15 ml centrifuge tubes. Theprecipitation layers remaining in the four tubes were collected into one50 ml centrifuge tube (FALCON) with a transfer pipette. Next, theprecipitation layers were suspended in 27 ml of ice-cooled distilledwater, so as to hemolyze the blood erythrocytes. Thirty seconds later, 3ml of a PIPES buffer at a 10-fold concentration, comprising 0.11M sodiumchloride, 5 mM potassium chloride, 25 mM piperazine-1,4-bis(2-ethanesulfonic acid) and 42 mM sodium hydroxide, was added to thetube, so as to reestablish isotonicity and stop the reaction. Then,centrifugation at 400×g for 5 minutes at 4° C. was carried out. Thesupernatant was decanted, and then the precipitate was well suspended in3 ml of a PIPES buffer. Then the similar procedure was repeated and theprecipitates in all the centrifuge tubes were transferred into one 15 mlcentrifuge tube, and then the precipitate was washed with 15 ml of aPIPES buffer containing 1% FCS (hereinafter referred to as 1%FCS-PIPES). The cell population was prepared as a granulocyte fraction(approximately 4×10⁷ to 6×10⁷ cells) and the number of cells wascounted. Subsequently, 2 μg of mouse anti-CD16 antibodies (Clone name:3G8, IMMUNOTECH) was added to 1×10⁷ cells. The antibodies and the cellswere allowed to react in ice with occasional stirring for 30 minutes.The mixture was washed twice by centrifugation with 1% FCS-PIPES, andthen sheep anti-mouse antibody-immobilized magnetic beads (DYNAL) in a4-fold volume of the number of cells were added to the mixture, followedby reaction in ice for 30 minutes with occasional stirring. Using amagnetic bead concentrator MPC-1 [DYNAL], the magnetic beads and CD16-expressing cell populations bound to the beads were removed. Only theremaining supernatant was transferred into a 15 ml centrifuge tube,centrifuged at 400×g for 5 minutes at 4° C., thereby collecting thecells. The number of cells in the pellet was counted, and the cellpopulation was used as an eosinophil fraction (approximately 1×10⁶ to2×10⁶ cells). The cells were prepared into specimens with a Cytospin(SHANDON), and then the specimens were stained with a Dif-Quick stain(INTERNATIONAL REAGENTS CORPORATION). Approximately 500 cells werecounted using a microscope so that the purity of eosinophils wascalculated. Eosinophils could be always isolated with a purity of 95% ormore.

EXAMPLE 2 Detection of Apoptosis of Eosinophils Based on Inhibition ofIL-5 Activity

The inhibition of survival of eosinophils was measured in vitro for eachantibody by measuring annexin V-positive apoptotic cells as shown below.The antibodies used herein were KM8399 and KM9399 (WO97/10354), whichare anti-human IL-5R α-chain antibodies; TRFK5 (Pharmingen), which is ananti-human IL-5 antibody; and KM8969, which is a human IgG1,anti-ganglioside GM2 monoclonal antibody as a control antibody (JapanesePatent Application Laying-Open (Kokai) No. 10-257893).

High purity (purity of 95% or more) eosinophils isolated by the methodof Example 1 were prepared at 2×10⁶ cells/ml with 1% FCS-RPMI. Then, 100μl of the prepared eosinophils was apportioned to each well of a 96-wellU-shaped plate.

It is known that Eosinophils prolong their lifetime in the presence of0.1 ng/ml rhIL-5 (R&D). Thus, 0.4 ng/ml rhIL-5 was apportioned at 50μl/well.

Each type of antibody was prepared at 4 μg/ml was apportioned at 50μl/well. In total, 200 μl of the prepared solution was cultured underthe air containing 5% CO₂, at 37° C. for 24 hours.

After culturing, the eosinophils in the wells were stained using anannexin V-FITC kit (TREVIGEN). Forward-scattered light (hereinafterreferred to as FS) and side-scattered light (hereinafter referred to asSS) were set on the fractions of the cells that have kept their cellforms. Then FL1 (the first fluorescence) was measured by a flowcytometer (Coulter), so as to calculate the proportion of annexin V-FITCpositive cell population to the total number of subject cells.Therefrom, the proportion of apoptosis-induced cells was obtained.Specifically, the proportion of apoptosis-induced cells was calculatedby taking rhIL-5-free annexin V-FITC positive % in the absence of rhIL-5as 100%, annexin V-FITC positive % in the presence of rhIL-5 and in theabsence of antibody as 0%.

As shown in FIG. 1, KM8399 and TRFK5 induced the apoptosis ofeosinophils based on inhibition of rhIL-5 activity to almost the samedegree. KM9399 (WO97/10354), which is a human IgG4 subclass having nocellular cytotoxicity, also has the similar activity. These resultssuggest that the apoptosis of eosinophils was induced by inhibiting IL-5activity.

EXAMPLE 3 Detection of Apoptosis of Eosinophils Induced by CellularCytotoxicity

(1) Detection of Apoptosis Induced by ADCC Using Effector Cells (I)

A 96-well U-shaped plate (FALCON) for cell cultivation was used. PBMCfractions from healthy volunteers, as prepared in above mentionedExample 1, were used as effector cells, and prepared at 1×10⁷ cells/mlwith 1% FCS—RPMI. The cell suspension was apportioned at 100 μl/well. Astarget cells, the fractions of eosinophils as prepared in Example 1 wereprepared at 8×10⁵ cells/ml, and then apportioned at 50 μl/well. At thistime, the effector/target ratio (E/T ratio) was approximated to be 25:1,which is similar to the ratio in vivo.

Next, 4 μg/ml KM8399 antibody diluent was added at 50 μl/well, and thenthe plate was allowed to stand under the air containing 5% CO₂ at 37° C.for 4 hours for ADCC reaction to proceed.

After reaction, media were removed from the wells, and then the cellswere stained using an annexin V-FITC kit (TREVIGEN) according to theinstruction attached to the kit, followed by analysis with a flowcytometer. FS and SS were set on the subject cells (the fractions of theeosinophils) in the samples, and then the fluorescence intensity of FL1was measured. Therefrom, the proportion of annexin V positive % toeosinophil fractions was calculated.

As shown in FIG. 2, when KM8399 was added, the annexin V-FITC positivecell population significantly increased, compared to the samples towhich antibody was not added, however, the PI positive cell populationdid not increase.

These results suggest that the mechanism of KM8399 to remove eosinophilsis induction of apoptosis.

(2) Detection of Apoptosis Induced by ADCC Using Effector Cells (II)

Detection was carried out in a similar manner to Example 3 (1) exceptthat the time of ADCC reaction was 20 hours. Apoptosis was examined bystaining with annexin V only. To measure with a flow cytometer based ondifferences between FS and SS, areas to be measured with thefluorescence intensity of annexin V were determined respectively forlymphocytes, monocytes and eosinophils. The annexin V positive % of eachsubject area was detected by the method described in Example 3 (1).

Added as antibodies were KM8399, the IgG1 type antibody having ADCCactivity; and KM9399, the IgG4 type antibody having almost no ADCCactivity. Added as control antibodies were KM871, the IgG1 type antibodyanti-ganglioside GD3 monoclonal antibody (Cancer Immunol. Immunother.,3, 373-380, 1993). These antibodies were all examined at a finalconcentration of 1 μg/ml.

As shown in FIG. 3, when KM8399, the IgG1 type antibody having ADCCactivity, was added, eosinophil-specific induction of apoptosis wasobserved, however, no reaction was found in the lymphocytes fraction andmonocytes fraction contained in the PBMC fraction. When KM9399, the IgG4type antibody having no ADCC activity, was added, no reaction wasobserved in any of the cells. These results suggest an ADCCactivity-related apoptosis induction mechanism by KM8399. KM871, theIgG1 type anti-ganglioside GD3 monoclonal antibody (Cancer Immunol.Immunother., 36, p373-380, 1993) as a negative control indicates nospecific activity.

(3) Comparison with Anti-IL-5 Antibody TRFK5

To determine whether the detected level of eosinophil apoptosisinduction by KM8399 as shown in Example (2) was significant, comparisonswith TRFK5 were examined using the PBMC fractions of 3 healthyvolunteers in a manner similar to the method of Example 3 (1).

As shown in FIG. 4, although apoptosis induction was observed whenKM8399 was added, no apoptosis induction was observed when TRFK5 wasadded.

A similar experiment carried out using the PBMC fractions of 3 healthyvolunteers yielded similar results.

Further, an experiment was carried out in a manner similar to the aboveexperiment using different concentrations of antibodies. The results areshown in FIG. 5. KM8399 showed an increase in apoptosis-inducingactivity in an antibody concentration-dependent manner, and a sustainedlevel of activity even at the final concentration of 0.01 μg/ml. Theseresults suggest that KM8399, the anti-human IL-5 receptor α-chainantibody having eosinophil-specific binding activity, possesses higherapoptosis-inducing activity, and is more preferred as a therapeuticagent for eosinophilic diseases, compared to TRFK5, the anti-human IL-5antibody.

(4) ADCC Activity in the Presence of Cytokines

ADCC activity was examined, when eosinophils were activated in thepresence of IL-5, IL-3 and/or GM-CSF, and then various antibody types (1μg/ml each) were acted upon the eosinophils.

PBMC fractions prepared at a 2-fold concentration of the PBMC fractionsused in Example 3(1) were apportioned at 50 μl/well; a 4 ng/ml diluentof each cytokine or a 12 ng/ml mixed solution of 3 types of cytokineswas apportioned at 50 μl/well; and then the fractions of eosinophils andantibody diluents were apportioned at 50 μl/well, similar to Example 3(1), so as to achieve the total volume of 200 μl/well. The cytokinesadded herein was either IL-5, IL-3 and/or GM-CSF (all the cytolinesmanufactured by R&D). The mixed solution of the cytokines contained theabove 3 types of cytokines (4 ng/ml).

As shown in FIG. 6, 1 μg/ml KM8399 significantly induced apoptosiscompared to the case that no antibody is added in the presence of anyone or all of the cytokines. In contrast, TRFK5 induced no apoptosis.

EXAMPLE 4 Measurement of Eosinophil Granular Protein

After ADCC reaction was carried out in a similar manner to Example 3(1), the plate was subjected to centrifugation at 350×g for 5 minutes at4° C., the supernatants were transferred into a 1.5 ml tube (Eppendorf),stored at 80° C., and then used as samples for quantitativelydetermining free eosinophil granular protein, as described below. Tomeasure all the granular proteins in the cells, 1% FCS-RPMI containing10% Triton X was added at 10 μl/well, cytolysis was performed, and thenthe culture supernatants were used as samples.

(1) Measurement of Eosinophil Peroxidase: EPO

50 μl of the culture supernatant sample was apportioned in duplicate toeach well of a 96-well ELISA plate (Greiner), and then a chromogenicsubstrate solution [solution consisting of 50 mM sodium citrate (pH5.0),0.4 mg/ml o-phenylene diamine and 30% hydrogen peroxide solution 1/1000]was added at 100 μl/well. The mixture was allowed to develop color for30 minutes. Subsequently, 4N sulfuric acid was added at 50 μl/well tostop color development, and then the absorbance at 490 nm was measuredusing a plate reader (Molecular Devices). As shown in FIG. 7, therelease % of EPO in the sample added with KM8399 was equivalent to thatin the sample to which no antibody was added.

(2) Measurement of Eosinophil-Derived Neurotoxin (EDN) 200 μl of anassay diluent contained in an EDN ELISA kit (MBL) was added to 50 μl ofthe culture supernatant following ADCC reaction, and then diluted5-fold, thereby preparing samples for measurement. The samples wereprepared in duplicate, and EDN was quantitatively determined accordingto the instructions of the kit. The absorbance was measured with a platereader (Molecular Devices), and the concentrations in the samples wereconverted based on a standard product in the kit using soft max(Molecular Devices). As shown in FIG. 8, the release % of EDN in thesample added with KM8399 was significantly lower, and showed nosignificant difference compared to that in the sample to which noantibody was added.

As described above, it could be confirmed that removal of eosinophils byKM8399 was caused by apoptosis, and not by necrosis in which cytoplasmssuch as EPO and EDN are fragmented and scattered. Specifically, wheneosinophils were removed by KM8399, no toxicity resulting from therelease of granular proteins within the eosinophils was observed toaffect the surrounding tissues or the cells.

EXAMPLE 5 Morphological Evaluation of Viability of Eosinophils AfterADCC Reaction

After ADCC reaction, 30 μl of a reaction solution containing the cellswas diluted 10-fold with 1% FCS-RPMI. 100 μl of the diluted solution wasapplied to each sheet of slide glass (SHANDON), and two specimens wereprepared with Cytospin for each sample. Similar to the evaluation of thepurity of eosinophils, specimens were stained with Dif-Quick stain(INTERNATIONAL REAGENTS CORPORATION). Next, 100 cells comprising viableand dead eosinophils (cells that nearly kept their forms) on each slideglass were counted under a microscope, thereby calculating theproportion of dead cells. As shown in FIG. 9, eosinophil-specificremoval resulting from the addition of KM8399 was confirmed, similar tothe result of apoptosis induction obtained using annexin V. Neither IgG4type KM9399 nor the negative control, IgG1 type anti-ganglioside GD3monoclonal antibody KM871 [Cancer Immunol. Immunother., 36, p373-380,1993], did not induce eosinophil-specific removal.

EXAMPLE 6 Concentration of CD16 Positive Cells in the Peripheral BloodGranulocyte

Granulocytes in peripheral blood consist of CD16 positive neutrophil andCD16 negative eosinophil fractions. Selective removal of eosinophilsfrom peripheral blood granulocytes was studied by measuring theproportion of CD16 positive cells by the following method.

Lymphocytes and granulocytes were fractionated by the method of Example1, and 50 μl of the fraction (5×10⁵ cells) was apportioned to each wellof a 96-well U-shaped plate. Then, KM8399 previously prepared at 2 μg/mlwith 10% FCS-RPMI was apportioned at 100 μl/well to the plate, and thencultured under the air containing 5% CO₂ at 37° C. for 96 hours. Afterculturing, cells were centrifuged at 350×g for 3 minutes at 4° C. toremove the supernatant. The cells were washed by adding 100 μl of abuffer for measurement [PBS (phosphate-buffered saline) containing 1%bovine serum albumin, 0.02% EDTA (ethylenediamine-N,N,N′N′-tetraaceticacid), 0.05% sodium azide] to each well. Then, FITC-labeled anti-CD16antibody (Nippon Becton Dickinson Company, Ltd.) was added at 20 μl/wellfor reaction to proceed on ice for 30 minutes. Then, the reactionproduct was washed by centrifugation with a buffer for measurement intriplicate, and then the fluorescence intensity of FITC was measuredwith a flow cytometer. The results are shown in FIG. 10.

In the group added with KM8399, CD16 negative eosinophils decreased andCD 16 positive neutrophils increased, compared to the group to which noantibody was added.

EXAMPLE 7 Detection of Apoptosis of Activated Eosinophils

The apoptosis detection method of 3 (1) mentioned above was improved,and then apoptosis of activated eosinophils was detected by the improvedmethod. 100 μl of eosinophils (4×10⁵ cells/ml) and of PBMC (1×10⁶cells/ml) isolated from peripheral blood were together added to eachwell of a 96-well plate, followed by co-culturing for 48 hours to induceactivated eosinophils. The thus co-cultured eosinophils expressed CD69molecules, the activated eosinophil marker.

After co-culturing, 100 μl of the culture supernatant was removed fromeach well, and then 100 μl of various antibody diluents (2 μg/ml) wasadded respectively (final concentration 1 μg/ml), followed by anotherculturing for 20 hours. After culturing, apoptotic cells were detectedby staining with annexin V-FITC.

As shown in FIG. 11, KM8399 significantly induced apoptosis of activatedeosinophils. Inhibiting IL-5 only cannot remove nor reduce activatedeosinophils, because activated eosinophils survive in the absence ofcytokines. These results suggest that KM8399 can remove or reduce notonly eosinophils in peripheral blood, but also activated eosinophilsinfiltrating inflammated areas, and that KM8399 is clinically useful.

INDUSTRIAL APPLICABILITY

The apoptosis inducer of the present invention is useful in treatinginflammatory disorders, such as chronic bronchial asthma, eosinophilicdiseases, such as eosinophilic granuloma, or the like, by inducingeosinophil apoptosis to reduce or remove eosinophils or activatedeosinophils.

1. A method for specifically inducing apoptosis of an eosinophil,comprising: administering an anti-human interleukin-5 receptor α-chainmonoclonal antibody with an Fc region of the human IgG1 subclass thathas antibody-dependent cellular cytotoxicity to a patient in needthereof in an amount effective to induce apoptosis of an eosinophil insaid patient.
 2. The method of claim 1, wherein the anti-humaninterleukin-5 receptor α-chain monoclonal antibody is produced by ananimal cell.
 3. The method of claim 1, wherein the anti-humaninterleukin-5 receptor α-chain monoclonal antibody is produced by thetransformant KM8399 (FERM BP-5648).
 4. A method for specificallyreducing or removing eosinophils in peripheral blood or in tissuesinfiltrated with eosinophils, compnsing: administering an anti-humaninterleukin-5 receptor α-chain monoclonal antibody with an Fc region ofthe human IgG1 subclass that has antibody-dependent cellularcytotoxicity to a patient in need thereof in an amount effective toremove or reduce said eosinophils.
 5. The method of claim 4, wherein theanti-human interleukin-5 receptor α-chain monoclonal antibody isproduced by an animal cell.
 6. The method of claim 4, wherein theanti-human interleukin-5 receptor α-chain monoclonal antibody isproduced by the transformant KM8399 (FERM BP-5648).
 7. The method ofclaim 1, wherein inducing apoptosis of said eosinophil treats chronicbronchial asthma, atopic dermatitis, eosinophilia, eosinophilicenterogastritis, eosinophilic leukemia, eosinophilic granuloma orKimura's disease in a patient in need thereof.
 8. The method of claim 2,wherein inducing apoptosis of said eosinophil treats chronic bronchialasthma, atopic dermatitis, eosinophilia, eosinophilic enterogastritis,eosinophilic leukemia, eosinophilic granuloma or Kimura's disease in apatient in need thereof.
 9. The method of claim 3, wherein inducingapoptosis of said eosinophil treats chronic bronchial asthma, atopicdermatitis, eosinophilia, eosinophilic enterogastritis, eosinophilicleukemia, eosinophilic granuloma or Kimura's disease in a patient inneed thereof.
 10. The method of claim 4, wherein removing or reducingsaid eosinophils treats chronic bronchial asthma, atopic dermatitis,eosinophilia, eosinophilic enterogastritis, eosinophilic leukemia,eosinophilic granuloma or Kimura's disease in a patient in need thereof.11. The method of claim 5, wherein removing or reducing said eosinophilstreats chronic bronchial asthma, atopic dermatitis, eosinophilia,eosinophilic enterogastritis, eosinophilic leukemia, eosinophilicgranuloma or Kimura's disease in a patient in need thereof.
 12. Themethod of claim 6, wherein removing or reducing said eosinophils treatschronic bronchial asthma, atopic dermatitis, eosinophilia, eosinophilicenterogastritis, eosinophilic leukemia, eosinophilic granuloma orKimura's disease in a patient in need thereof.